Genetically engineered negative signaling molecules in the
immunomodulation of allergic diseases
Andrew Saxona, Daocheng Zhua, Ke Zhanga, Lisa Chan Allena and
Christopher L. Kepleyb
Purpose of review
This review summarizes current knowledge regarding the
control of human mast cell and basophil signaling and recent
developments using a new therapeutic platform consisting of a
human bifunctional g and e heavy chain (Fcg–Fce) protein to
inhibit allergic reactivity.
Recent findings
Crosslinking of FcgRIIb to FceRI on human mast cells and
basophils by a genetically engineered Fcg–Fce protein (GE2)
leads to the inhibition of mediator release upon FceRI challenge.
GE2 protein was shown to inhibit cord blood-derived mast cell
and peripheral blood basophil mediator release in vitro in a
dose-dependent fashion, including inhibition of human IgE
reactivity to cat. IgE-driven mediator release from lung tissue
was also inhibited by GE2. The mechanism of inhibition in mast
cells included alterations in IgE-mediated Ca2+ mobilization,
spleen tyrosine kinase phosphorylation and the formation of
downstream of kinase–growth factor receptor-bound protein 2–
SH2 domain-containing inositol 5-phosphatase (dok–grb2–
SHIP) complexes. Proallergic effects of Langerhan’s like
dendritic cells and B-cell IgE switching were also inhibited by
GE2. In vivo, GE2 was shown to block passive cutaneous
anaphylaxis driven by human IgE in mice expressing the human
FceRI and inhibit skin test reactivity to dust mite antigen in a
dose-dependent manner in rhesus monkeys.
Summary
The balance between positive and negative signaling controls
mast cell and basophil reactivity, which is critical in the
expression of human allergic diseases. This approach using a
human Fcg–Fce fusion protein to co-aggregate FceRI with the
FcgRII holds promise as a new therapeutic platform for the
immunomodulation of allergic diseases and potentially other
mast cell/basophil-dependent disease states.
Keywords
allergic reactions, FceRI, FcgRIIb, mast cells and basophils,
negative signaling transduction
Curr Opin Allergy Clin Immunol 4:563–568.
#
2004 Lippincott Williams & Wilkins.
a
The Hart and Louise Lyon Laboratory, Division of Clinical Immunology/Allergy,
Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles,
California, and bDivision of Rheumatology, Allergy and Immunology, Department of
Internal Medicine, Medical College of Virginia, Richmond, Virginia, USA
Correspondence to Andrew Saxon MD, Professor and Chief, 52-175 CHS, Division
of Clinical Immunology, Department of Medicine, UCLA School of Medicine, 10833
LeConte Ave, Los Angeles, CA 90095-1680, USA
Tel: +1 310 206 8050; fax: +1 310 206 8107; e-mail: [email protected]
Abbreviations
CBMC
Dok
Grb2
ITAM
ITIM
LLDC
PCA
SHIP
SHP
Syk
NIP
FceRI
FcgRII
cord blood-derived mast cell
downstream of kinase
growth factor receptor-bound protein 2
immunoreceptor tyrosine-based activation motif
immunoreceptor tyrosine-based inhibition motif
Langerhan’s like dendritic cell
passive cutaneous anaphylaxis
SH2 domain-containing inositol 5-phosphatase
SH2 domain-containing phosphatase
spleen tyrosine kinase
4-hydroxy-3-iodo-5-nitrophenylacetyl
high-affinity receptor for IgE Fc
receptor II for IgG Fc (CD32)
#
2004 Lippincott Williams & Wilkins
1528-4050
Introduction
Mast cell and basophil mediator release driven by the
immunoreceptor
tyrosine-based
activation
motif
(ITAM)-containing FceRI is a critical component in
expression of human immediate hypersensitivity-allergic
disease. For years, various pharmacologic approaches
have been employed to antagonize the effects of these
mediators [1 .,2]. More limited success has been
achieved with approaches aimed at inhibiting mediator
release [3]. A great deal of new information about the
role of negative signaling in mast cells and basophils via
immunoreceptor tyrosine-based inhibition motif (ITIM)containing receptors has opened the way for new
approaches that employ negative signaling as a way of
controlling human allergic reactions [4,5]. Negative
regulation of signaling through ITAM-containing receptors by signals from co-crosslinked ITIM-containing
FcgRIIb has been demonstrated in mouse and human
cells [6–8]. The ability of the ITIM-containing IgG
receptor FcgRIIb to act as a negative regulator of ITAMcontaining immunoreceptors was recognized initially
through studies of the antigen receptor of B cells and
was subsequently extended to studies of T-cell activation through T-cell receptors [9]. FcgRIIb was first
implicated in the negative regulation of human FceRImediated signaling by Daeron et al. [6]. It was later
extended to peripheral blood basophils [8] and cord
blood-derived human mast cells (CBMCs) [10 . .]. Herein
we review current progress in understanding the
regulation of human mast cells and basophil signaling,
and highlight a potential therapeutic approach using a
novel human Fcg–Fce fusion protein to achieve inhibition of allergic reactivity.
Current Opinion in Allergy and Clinical Immunology 2004, 4:563–568
563
564 Immunotherapy
Inhibitory signaling in the control of human
basophils and mast-cell function
The goal of immunotherapy is to induce mast cell/
basophil nonresponsiveness by injecting suboptimal
concentrations of allergen to induce mast cell/basophil
inhibition. However, it is not known why mast cells and
basophils become desensitized after this treatment. One
hypothesis proposes that ‘blocking antibodies’ leads to
co-aggregation of FcgRIIb receptors with FceRI. During
immunotherapy the serum levels of antigen-specific IgG
levels increase such that antigen-specific IgG (complexed to Ag or possibly as monomeric IgG) binds to
FcgRIIb on mast cells and basophils. Upon subsequent
allergen exposure, FceRI-bound IgE binds to IgG
complexed to antigen, leading to co-aggregation of
FceRI and FcgRIIb that initiates an inhibitory rather
than an activating signal.
Daeron et al. [6] were the first to demonstrate that FceRI/
FcgRII co-aggregation on human basophils inhibited
IgE-mediated degranulation through ITIM signaling.
We extended these studies in a more biologically
relevant model system, which showed that antigeninduced basophil histamine release and IL-4 production
could be inhibited by co-aggregation of Fce–Fcg using
IgE with IgG to the same aggregated antigen [8]. It is
not clear, however, if this mechanism occurs in vivo.
GE2, a human Fcg–Fce fusion protein, inhibits
human basophil and mast cell function in
vitro
Realizing the therapeutic potential for regulating FceRI
signaling, we developed a molecule that utilizes FceRI
co-aggregation to ITIM-containing receptors [11]. This
fusion protein (GE2) consists of the human IgG1 g
hinge–CHg2–CHg3 region linked to the human IgE
CHe2–CHe3–CHe4 region. The human g1 hinge, CH2–
CH3 region was placed ahead of the e heavy chain CH2–
CH4 domains with a 17-amino-acid linker containing a
BgIII site and (Gly4Ser)3 positioned between the two Fc
regions. This linker facilitates chain pairing and minimizes refolding and aggregation problems encountered
when the two chains are expressed individually [12].
GE2 was characterized as the predicted *140 KDa
dimer and contained the binding sites for FcgRII
(CHg2–CHg3) and FceRI (CHe2–CHe3) [13,14]. The
first constant domains (CHg1 and CHe1) were deleted as
these domains associated with BiP (immunoglobulin
heavy chain binding protein). GE2 does not contain light
chains [15]. The proposed binding and mechanism for
GE2’s inhibitory effects on human mast cells and
basophils is shown in Fig. 1.
Human basophils purified from the peripheral blood,
known to express FcgRIIb were passively sensitized
with 10 mg/ml of chimeric human anti-4-hydroxy-3-iodo-
5-nitrophenylacetyl (NIP) IgE. One hour later, doses of
GE2 ranging from 0.01 to 10 mg/ml were added and
compared with a human IgE myeloma control. After
another hour of incubation, the cells were activated with
antigen NIP-BSA. At 10 mg/ml of GE2, there was an
average of 84% inhibition while 1 mg/ml of GE2
inhibited histamine release by about 59%. Myeloma
IgE at 10 mg/ml only decreased the release by 19%.
Similarly, when naturally sensitized basophils obtained
from cat-allergic individuals were incubated with GE2
and then challenged with purified cat antigen, Fel D1,
GE2 again showed a dose response with 78% inhibition
of Fel D1 specific release at 10 mg/ml of GE2 [16 . .].
Importantly, GE2 itself did not cause degranulation
when it was incubated with basophils sensitized in vivo
to cat. GE2’s effects on basophil mediator release were
also time dependent; as the time between GE2 addition
and the stimulus for release was decreased, so was GE2’s
ability to inhibit. In addition, we have shown that GE2
can inhibit mast cell/basophil function from human lung
tissue that had bound IgE in vivo. Recently, we
demonstrated that CBMC, IgE-mediated responses
could be inhibited with GE2 (Fig. 2). Therefore, blood
basophils and CBMCs express the ITIM-containing
FcgRIIb isoform which, when co-aggregated to FceRI,
inhibits IgE-mediated release. Taken together, these
differing approaches show that GE2 can inhibit IgEmediated mediator release in passively or natural
sensitized human basophils and mast cells.
GE2 functions to inhibit mast cell and
basophil signaling
The mechanisms of inhibition through FceRI–FcgRII
co-aggregation highlight the differences between the
human and rodent systems. First, rodent studies using
cell lines implicate SH2 domain-containing inositol 5phosphatase (SHIP) more so than SH2 domain-containing phosphatase (SHP-1) in FcgRII-mediated inhibitory
signaling [17–19]. In contrast, our data with peripheral
blood basophils implicate SHP-1 in the FcgRIImediated inhibitory signaling [11,20]. Second, in rodents, co-aggregation of FceRI to FcgRII does not
prevent FceRI-mediated activation of spleen tyrosine
kinase (Syk) [7]. However, we have shown in peripheral
blood basophils [8,11] that FcgRII appears to regulate
some part of Syk function as has been demonstrated in
other human cells [21 .]. Third, while FceRI–FcgRII coaggregation in rodent and human systems inhibits the
Ca2+ response to FceRI stimulation, we have demonstrated that the kinetics of this inhibition is distinct in
peripheral blood basophils [8] and CBMCs [10] compared with rodent cells [17,18]. Finally, in results not
observed in rodent systems, co-aggregation in human
systems increased the tyrosine phosphorylation of the
adapter protein downstream of kinase (Dok), growth
factor receptor-bound protein 2 (Grb2), and SHIP.
Immunomodulation of allergic diseases Saxon et al. 565
Figure 1. Schematic showing the proposed mechanism for the genetically engineered Fcg–Fce protein (GE2) effect on human basophils and
mast cells
(a) Antigen crosslinking two FceRIs and inducing
activation with enhancement of spleen tyrosine
kinase (Syk) and other downstream signaling
molecules. FcgRII is unoccupied. (b) GE2
co-crosslinking FcgRIIb to FceRI, which results
in the inhibition of signaling events downstream
of the FceRI immunoreceptor tyrosine-based
activation motif (ITAM), such that the cell is
inhibited in its ability to function via FceRI
activation. ITIM, immunoreceptor tyrosine-based
inhibition motif.
(a) No FcεR1/FcγRII co-crosslinking
Normal ITAM-mediated signaling
via Syk phosphorylation
FcεR1/FcγRII co-crosslinking
ITAM-signaling inhibited
(b)
ITIM
ITAM
Ag
IgE
GE2
GE2
IgE
Fcγ RII
FcεR1 (2)
Increased Syk
phosphorylation
Inflammatory mediator
release
Tyrosine phosphorylation of Dok was associated with
increased binding to Grb2. Surprisingly, in nonstimulated cells, there were complexes of phosphorylated
SHIP–Grb2–Dok, which were lost upon IgE-receptor
activation but retained under conditions of FceRI–
FcgRII co-aggregation. Our results implicate Dok, SHIP,
and Grb2 as key intermediates in regulating IgEmediated degranulation and cytokine production [10 . .].
They further implicate these signaling intermediates as
the true ‘gatekeepers’ [22] of human mast cell degranulation. Clearly, the results from rodents cannot be
directly extrapolated to the situation in humans.
FceR–FcgR co-aggregation in vitro inhibits
key proallergic processes in human B cells
and Langerhan’s like dendritic cells
While basophils and mast cells were the primary targets
for which GE2 was designed, other cells express both
FcgRII and FceRs, for example human Langerhan’s
like dendritic cells (LLDCs) express FceRI and FcgRII
while human B cells express FcgRII and FceRII
(CD23), the low-affinity IgE receptor. Thus we have
tested the ability of GE2 to inhibit the function of
those cells in vitro through receptor co-aggregation.
Notably, an antibody to CD23 has been shown to
inhibit isotype switching to IgE by human B cells [23 .].
We have shown that GE2 is extremely potent at
inhibiting various steps involved in IL-4 plus CD40driven class switch recombination and subsequent IgE
Mast cell or
basophil
Syk phosphorylation
inhibited
Inflammatory mediator
release inhibited
production. The resulting inhibition resulted from a
combination of inhibition of initiation of e germ line
transcription plus a direct effect on the process of the
isotype switch itself. The effects of GE2 on class switch
recombination in human B cells were clearly shown to
require binding to both CD23 and FcgR. This
inhibition of class switch recombination was dependent
on CD23 binding and the phosphorylation of extracellular-signal related kinase, and it was mediated via
suppression of IL-4-induced STAT6-phosphorylation
[24 . .].
Unstimulated LLDCs derived from CD14-positive
monocytes from atopic donors were shown to express
FcgRII. When passively sensitized with antigen-specific
human IgE and then challenged with antigen, LLDCs
were stimulated to produce IL-16, an important proallergic cytokine. IL-16 enhances the production of
inflammatory mediators such as histamine, RANTES
and monocyte chemotactic protein-1 and is a potent
chemoattractant for CD4+ T cells into areas of allergic
inflammation. However, when FceRI and FcgRII were
co-aggregated with GE2, IL-16 production was significantly inhibited in a dose-dependent fashion up to 63%.
Exposure of LLDCs to GE2 alone did not induce IL-16
production [25 .]. These results further extend our
studies demonstrating the ability of GE2 to inhibit
FceRI-mediated responses through co-aggregation with
FcgRIIb and at the same time show that human LLDCs
566 Immunotherapy
Figure 2. FcgRII/FceRI co-aggregation inhibits FceRI-mediated mast cell degranulation and tumor necrosis factor a production
4-Hydroxy-3-nitrophenylacetyl-IgE-sensitized
cord blood-derived mast cells (CBMCs) were
incubated with or without genetically engineered
Fcg–Fce protein (GE2) or nonspecific IgE (PS).
The cells were washed, incubated with or
without 200 ng/ml 4-hydroxy-3-iodo-5nitrophenylacetyl (NIP)-BSA or anti-IgE for
45 min (histamine release) or 12 h (cytokine
production) and mediator release measured in
the supernatants. Results are represented as the
standard error of the mean of four (a) or two (b)
separate experiments (+SEM). *Indicates
values significantly reduced (P50.01) when
comparing cells challenged with or without GE2.
(Previously published in Kepley et al. [10 .].)
50
(a)
Percent
histamine
release
40
P
*P=0.04
30
20
10
0
IgE anti-NIP
PS IgE (10 µg/ml)
GE2 (µg/ml)
NIP-BSA (200 ng/ml)
anti-IgE (1 µg/ml)
+
–
–
–
–
+
–
–
+
–
+
–
0.05
+
–
(b)
pg TNFα 50
(106 mast
cells)
+
–
0.1
+
–
+
–
0.5
+
–
+
–
1
+
–
+
–
10
+
–
+
+
–
+
–
–
+
–
–
+
+
–
5
+
–
+
–
10
+
–
+
+
–
+
–
–
+
–
–
+
P
*p<0.001
P
*p=0.004
40
30
20
10
0
IgE anti-NIP
PS IgE (10 µg/ml)
GE2 (µg/ml)
NIP-BSA (200 ng/ml)
anti-IgE (1 µg/ml)
can be modulated in a fashion similar to mast cells and
basophils.
GE2 inhibits basophil and mast cell function
in vivo
We have tested the ability of GE2 to function in vivo to
inhibit allergic reactivity through mast cells and basophils. In the first approach, we employed transgenic mice
that express the human FceRIa and have the murine
+
–
–
–
–
+
–
–
+
–
+
–
0.1
+
–
+
–
0.5
+
–
+
–
1
+
–
FceRIa chain knocked out, hFceRIatg mice. The human
FceRIa chain combines with the mouse b and g chains to
form a functional chimeric human/mouse FceRIa receptor. These animals demonstrate allergic reactivity
mediated by passively administered human IgE antibody when challenged with antigen. At the same time,
the murine FcgRIIb expressed on these hFceRIa
transgenic animals’ mast cells binds human IgG so that
GE2 is expected to be functional in them.
Immunomodulation of allergic diseases Saxon et al. 567
GE2 blocked passive cutaneous anaphylaxis at
sensitized skin sites in human Fce–RIa transgenic mice
We utilized passive cutaneous anaphylaxis (PCA) in the
hFceRIatg mice to test the ability of GE2 to block IgEdriven in vivo human FceRI-mediated mast cell release.
hFceRIatg mice were locally sensitized at multiple sites
with 250 ng of model genetically engineered human IgE
antibodies to 4-hydroxy-3-nitrophenylacetyl or dansyl.
Varying doses of GE2 or control myeloma IgE was
added at the sites. The mice were then challenged
intravenously 6 h later with the relevant antigen bound
to a carrier and reactivity was assessed. GE2 was able to
block PCA reactivity in a dose-dependent fashion, with
complete inhibition occurring at 250 ng of GE2.
These experiments were extended using human serum
containing high levels of IgE to cat. FceRIa transgenic
mice were locally sensitized at individual skin sites with
serum from a highly cat-allergic individual. Four hours
later, GE2 or control material was administered to these
sites and a further 4 h later, the mice were challenged
intravenously with Fel d1 and Evans blue dye.
Reactivity was scored 15–20 min later. PCA reactivity
was inhibited in a dose-dependent manner by GE2
protein, injected with complete inhibition at 2 mg.
Treatment of the cat-allergic serum at 568C for 30 min,
conditions known to abolish IgE binding to FceRI,
abolished PCA reactivity, thereby showing that the Fel
d1-specific IgE was responsible for the PCA reactivity.
Reactivity was not significantly blocked by administration of equivalent amounts of human IgE or human IgG.
Importantly when an intravenous challenge of Fel d1
was given immediately after administration locally of
only GE2, there was no local reaction, showing that GE2
itself was not causing the reactions at the injection sites.
GE2 markedly inhibited skin test reactivity to dust mites
in rhesus monkeys
Rhesus monkeys express skin test reactivity and serum
IgE directed toward dust mites [26]. We tested whether
GE2 could inhibit Dermatophagoides farinae mite skin test
reactivity in rhesus monkeys. D. farinae reactive monkeys underwent graded intradermal injections with GE2
(62.5–250 ng) versus purified human IgE myeloma
protein as a control. Five hours later, the sites were
then challenged with D. farinae at a dose optimized for
reactivity for each animal through previous dose–
response testing. Animals were simultaneously given
Evans blue dye intravenously and skin test reactivity was
measured 15–20 min later. GE2 protein effected complete inhibition at 250 ng and showed complete inhibition in four of five animals at 125 ng. At 62.5 ng, GE2
still exhibited clear inhibition of reactivity compared
with saline or control, while nonspecific IgE did not
show significant inhibitory effects on skin test reaction in
any of the doses tested. These results clearly indicate
that GE2 protein is able to inhibit naturally occurring
dust mite allergen induced allergic skin reactivity in
nonhuman primates in a dose-dependent fashion.
Future directions, enhancing FcgRII-related
effects and utilization of alternative inhibitory
signaling targets
The function of GE2 may be improved by increasing its
affinity to FcgRII relative to other FcgRs. This is being
undertaken by making mutations in key sites known to
alter FcgR binding. Optimizing the linker length
between the Fce and Fcg may provide enhanced avidity
of the molecule. Using the flexible hinge of the g1 chain
as the linker through reversing the order of the GE2
construct also may enhance function. Recently, an
approach using bi-specific, murine antibodies to achieve
co-aggregation of FcgRII and FceRIs has also been
shown to work on human basophils and cultured mast
cells in vitro [27 .].
There are several ITIM-containing receptors that have
been identified, but their expression has not been
investigated on human FceRI-positive cells [28]. We
investigated the expression of several ITIM-containing
receptors on peripheral blood basophils, CBMCs, the
human mast cell line, and the human basophil cell line.
As seen in Table 1, peripheral blood basophils and
CBMCs express only the ITIM-containing FcgRIIb
isoform of FcgRII. Thus, future strategies aimed at
harnessing the inherent inhibitory ability of these
Table 1. Immunoreceptor tyrosine-based inhibition motif (ITIM) receptor expression in human mast cells and basophils
Basophils
CBMCs
HMC-1
KU812
FcgRII
FcgRIIA
FcgRIIB
MAFA
Siglec-5
SIRP-a
CD 22
CD81a
LIR
+++
++
0–+
++
0–+ DV
0
0
0
+++
++
0–+
0–+
0–+
0–+
0
0
0
0–+++b
0
0
0
0–+++c
0
0
0
+–+++d
0
0
0–+ DV
+++
0
0
++–+++ DV
+
0
0
Detection of ITIM receptors on human FceRI-positive cells using anti-ITIM receptor antibodies and utilizing the methodology as described previously [29].
Peripheral blood basophils (490%), cord-blood derived mast cells (CBMCs), the human mast cell line (HMC-1), and the human basophil cell line
(KU812) were tested. The numbers represent approximate fluorescent intensity with +++ being the strongest and 0 representing no reactivity. NKG2,
KIR, CD5, 31, 32, 72 were not detected on any of these cell populations. DV, donor variation. In human blood basophils we found donor variation
expression of the FcgRIIA isoform and the leukocyte immunoglobulin-like receptor (LIR). aThe CD81 contains an ‘ITIM-like’ sequence. b–dIn CBMCs, IFNgdramatically upregulated siglec-5 (b) and downregulated CD22 (d), while IgE and IL-4 upregulated the signal-regulatory protein a (SIRP-a, c).
568 Immunotherapy
receptors targeted specifically to mast cells and basophils
may have therapeutic potential.
11 Zhu D, Kepley CL, Zhang M, et al. A novel human immunoglobulin Fcg-Fce
bifunctional fusion protein inhibits FceRI-mediated degranulation. Nat Med
2002; 8:518–521.
Conclusion
12 Houston JS, Levinson D, Mudgett-Hunter M, et al. Protein engineering of
antibody binding sites: recovery of specific activity in an anti-digoxin singlechain Fv analogue produced in Escherichia coli. Proc Natl Acad Sci U S A
1988; 85:5879–5883.
Crosslinking of FceRI and FcgRIIb induces strong
inhibitory signaling in CBMCs and peripheral blood
basophils. We have shown that this can be accomplished
with clear antiallergic effects in vitro and in vivo with a
single human Fcg–Fce fusion molecule. The ability of
GE2, in addition, to block human B-cell isotype switching to e and to inhibit proallergic function in LLDCs
suggests GE2 may provide broader benefits in the
treatment of IgE-mediated diseases than simply through
inhibition of mediator release. Thus, the approach of
using a chimeric Fcg–Fce fusion protein provides a
platform for antigen nonspecific immunomodulation of
allergic diseases. The work accomplished so far lays a
strong foundation for the development of this platform
as a human therapeutic intervention. Modifications in
the GE2 platform, such as mutations that increase
binding to FcgRII or decrease potential binding to
FcgRI/III, or alterations in the linker length that increase
co-aggregation, are now being explored.
Acknowledgements
Supported by USPHS grants AI-15251 and ongoing support from the
Bridges Larson Foundation. CLK was additionally supported by a grant
from the American Lung Association and AD Williams Foundation.
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